Recent application of algal culture for the production of ... · Acetone ACN DCM HP-20 AZA1 + 2 (µg·g−1) 17.4 ± 0.5 18 ± 2 17 ± 1 17 ± 1 Purity (%) 0.036 ± 0.002 0.07 ±

Recent application of algal culture for the production of

azaspiracids

& recent initiatives for the integration of environmental

and productive science with commercial stakeholders

Thierry Jauffrais & Philipp Hess

Ifremer, Laboratoire EMP/PHYC, France

Contact: [email protected]

AZA1-2

AZA1 and -2

4th “Rendez-vous de Concarneau : Where Industry meets Science in Marine Biotechnology”, August 2012

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Introduction

Short history of a discovery: 1995: Human intoxication in the Netherlands with Irish mussels

Symptoms are similar to DSP (Nausea, stomach cramp, diarrhoea)

1998: Azaspiracid-1 isolated by Satake et al.,

2003: Structure revised by Nicolaou et al. ,

Protoperidinium crassipes producer of AZAs? (James et al.)

2007-2008: First isolation (3D9) Near Scotland (2007) then in Denmark (UTH E2, 2008) (Krock et al., 2009)

2009: Description of Azadinium spinosum (Tillmann et al.)

2010: Description of Azadinium obesum (Tillmann et al.)

2011: Description of Azadinium poporum (Tillmann et al.)

Tillmann et al., 2009

Introduction Materials and methods Results Conclusion Objectives

AZA1 and -2


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Azaspiracid

Azadinium sp.

Azaspiracids shellfish poisoning

Localisation



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Azaspiracids

AZA1-2 : A. spinosum

AZA3-32: Metabolites,

artefacts & postulated AZAs



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Objectives

Observations:

Limited amount of AZAs available for => Toxicological studies

=> Instrument calibration in monitoring programs

Objectives:

Development of AZA1 and -2 production without having to rely on natural events

=> Pilot scale culture of A. spinosum

=> Development of extraction procedures from large culture volume

=> Improvement of the isolation procedure



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Materials and methods

Production system



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• Two 100L stirred photobiorectors

• Harvesting tank 300 L

• Filtered sea water

• pH: 7.9

K modified medium (Keller et al., 1987)

• Photocycle (16L/8D)

• PFD: 200 µmol phot.m-2.s-1

• Temperature: 18

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C

• Different dilution rate : 0.15, 0.2, 0.25, 0.3 day-1

Culture conditions

Harvesting procedure

• Tangential flow filtration (Sartorius Stedim Biotech)

• 5

0.1m² open-channel microfiltration cassette

•

30-50 L.h -1

• Continuous centrifugation (Clara 20, Alfa Laval)

• 70 L.h-1

• 11 000 g



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Extraction procedure

The algal concentrate:

Method 1.

Centrifugtion (3500 g, 30 min, 4 °C)

3 succesive extractions (Organic solvants tested: Acetone, ACN, DCM)

Evaporation and reconstitution in 5 mL MeOH

Method 2.

Sonication

25 g of activated Diaion HP-20 polymeric resin

24 h of contact time on a laboratory shaker

Elution with three volumes of acetone (50 mL) at 1 mL·min −1

Evaporation and reconstitution in 5 mL MeOH

Permeate:

Method 3.

Eight SPATT bags (8 × 3 g resin) + submerged pump

72 h of contact

Extraction as method 4

Method 4.

A submerged pump (20 L·min−1) + column (25 g resin)

72 h of contact

extracted as method 4



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Isolation procedure

Ethyl acetate

70%

EtOAc/MeOH

AZA1: Fr 24-34

AZA2: Fr 35-45Step 4

Flash-phenyl hexyl

1M NaCl

Hexane

100% EtOAc

90% EtOAc/MeOH

50% EtOAc/MeOH

100% MeOH

Purified AZAsStep 5

Prep HPLC-C18/C8

Step 1

Extraction

Step 2

Partitioning 1

Step 3

Silica gel

Ethyl acetate

70%

EtOAc/MeOH

AZA1: Fr 24-34

AZA2: Fr 35-45Step 4

Flash-phenyl hexyl

1M NaCl

Hexane

100% EtOAc

90% EtOAc/MeOH

50% EtOAc/MeOH

100% MeOH

Purified AZAsStep 5

Prep HPLC-C18/C8

Step 1

Extraction

Step 2

Partitioning 1

Step 3

Silica gel

Step 1

Extraction

Step 2

Partitioning 1

Step 3

Silica gel



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Results

R1 R2 R1 R2 R1 R2 R1 R2

AZA1 + 2 (fg·cell−1) 67 ± 3 98 ± 5 44 ± 13 95 ± 16 38 ± 2 86 ± 3 24 ± 1 63 ± 5

Toxin production AZA1 + 2

(µg·day−1)193 ± 9 314 ± 15 170 ± 50 406 ± 64 180 ± 10 475 ± 17 134 ± 5 415 ± 33

5.6 ± 0.2 6.6 ± 0.1Cell production (×109 cell·day−1)

Concentration (×103 cell·mL−1)

2.90 ± 0.09 3.21 ± 0.05 3.9 ± 0.2 4.3 ± 0.1 4.8 ± 0.2 5.5 ± 0.1

221 ± 5 187 ± 5 220 ± 4

0.3 day−1

193 ± 6 214 ± 3 194 ± 8 214 ± 7 190 ± 6

A. spinosum 0.15 day−1 0.2 day−1 0.25 day−1

Cell concentration stable at the different steady states tested

AZAs concentration decrease when flow rate increase

Toxin optimum of productivity around 0,25 day -1 (25L/day) under the

the studied conditions



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Acetone ACN DCM HP-20

AZA1 + 2 (µg·g−1) 17.4 ± 0.5 18 ± 2 17 ± 1 17 ± 1

Purity (%) 0.036 ± 0.002 0.07 ± 0.01 0.09 ± 0.01 0.21 ± 0.03

Final yield ~ 80%

Extraction recovery

Method No. Method description % Recovery of total

1 Algal paste 56 ± 9

2 Algal retentate + HP-20 54 ± 3

3 Algal permeate + SPATT 21 ± 9

4 Algal permeate + SPE 26 ± 4

Extraction : HP20 vs organic solvants

Same extraction yield

Better purity using HP20



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11.5 mg AZAs -1200 L of culture

4 isolation steps

Purity >95%

Isolation recovery

Step No. Step AZA1 (mg) AZA2 (mg) Weight (g) Purity (%)

HP-20 resin extract 12.5 3.2 3.04 0.5

1 Partitioning 11.2 3 1.32 1.1

2 Silica gel 10.2 2.8 0.17 7.6

3 Flash (Phenyl-Hexyl) * 9.7 2.4 0.01 >90

4 Prep HPLC (C8/C18) 9.3 2.2 - >95

% Recovery (steps 1–4) 75 70

* AZA1 and AZA2 were separated from each other in this step



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Conclusion

• A. spinosum is able to grow in pilot scale photobioreactors

at an interesting AZA concentration

• Development of reliable extraction procedures was possible

• Simplification of the isolation procedure was achieved



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Prices of AZAs, NRC/cifga:

AZA1 : 1µg =

160€

AZA2 : 1µg =

260-280€

9.3 mg AZA1 =

1 488 K€

2.2 mg AZA2 =

572K€

Galla

rdo

-Ro

drig

uez

2012



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2

Recent initiative integrating environmental & productive

science with societal & commercial stakeholders: COSELMAR

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A key project of the research federation IUML

Partners : Université de Nantes, Ifremer + other universities

(Paris, Montpellier, Poitiers, CA, BE, DE)

Regional fiancial support (Pays de la Loire): 2.1 Mio €

Comprehension of Coastal and Marine Socio-

Ecosystems for the improved Exploitation of Marine

Resources, and better Prevention and Management of

Risks (COSELMAR)

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2 COSELMAR: Comprehension of coastal & maritime socio-ecosystems

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Socio-economic Partners

Atlanpôle

Pôle Mer Bretagne

Chamber of Commerce & Industry Nantes / St Nazaire

(Comité Consultatif Régional)

Maison des Sciences de l’Homme (MSH)

Industrial Partners

► BioLittoral (Micro-entreprise): monitoring

► (Shell-) fishermen (SME): sampling

► STX (Multinational): biofouling & maritime structures

► Agilent (Multinational): chemodiversity of toxic µ-algae

Chemical diversity of toxic µ-algae - dereplication

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V. rugosum – bioguided fractionation

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Bioguided fractionation V. rugosum

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VulcanodiniumEB DCMMeOHaq SiO2 F3

Pinnatoxin-G

Also, a number of compounds initially identified in sponges can be

attributed now to Vulcanodinium rugosum !

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Bioguided fractionation V. rugosum

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Thank you for your attention

5 µm


Documents

Recent application of algal culture for the production of ... · Acetone ACN DCM HP-20 AZA1 + 2 (µg·g−1) 17.4 ± 0.5 18 ± 2 17 ± 1 17 ± 1 Purity (%) 0.036 ± 0.002 0.07 ±